It is known that X-ray microscopes can perform high-resolution observation of a sample in a solution, giving a useful technique in the case where the observation target sample is a biological sample particularly. Particularly, on X-rays in a wavelength range of 2.3 to 4.4 nm (corresponding to an energy range of 284 to 540 eV) generally called “water window” (soft X-rays), differences in absorption coefficient between the constituent substances of the living body are large and the relevant X-rays, while transmitted without being absorbed by water, are liable to be absorbed by carbon and nitrogen, this leading to characteristics in which proteins and the like hardly allow the relevant X-rays to be transmitted. X-ray microscope observation using the soft X-rays is suitable for observing a biological sample.
The above-mentioned characteristics that the soft X-rays in the wavelength region of “water window” have enable an observation target object containing moisture (biological sample or sample in a solution) to be observed as it is, and in addition, enables observation in higher resolution than in the optical microscope observation because of the shorter wavelength than that of the visible light. Such reasons are promoting development of the microscope utilizing the X-rays in the “water window” wavelength region (soft X-ray microscope).
Notably, soft X-rays in a wavelength region of “carbon window” (5.0 to 4.5 nm) showing less absorption by carbon and soft X-rays in a region of a further shorter wavelength (0.6 to 2.3 nm) are also effective for observing a biological sample.
X-ray microscopes are categorized mainly into a scheme of irradiating the sample with an X-ray beam thinly focused using a light collecting system such as a zone plate (light collecting scheme) and a scheme of irradiating the sample with an X-ray beam from a point light source (point light source scheme).
The X-ray microscopes in the light collecting scheme are categorized into ones of irradiating transmission type and ones of scanning transmission type. The resolution depends on processing precision of the zone plate and the like and its theoretical limit is predicted to be approximately 10 to 15 nm.
On the other hand, the X-ray microscopes in the point light source scheme are categorized into a scheme of generating X-rays with a laser and a scheme of generating X-rays by means of irradiation with a charged particle beam such as an electron beam. There are proposed examples of the scheme of generating X-rays by means of irradiation with a charged particle beam, including a technique that an electron beam is allowed to incident on and through the surface of a sample supporting film to generate the X-rays in the sample supporting film and samples adhering to the rear face of the sample supporting film is irradiated with the X-rays (Patent Literature 1: Japanese Patent Laid-Open No. 8-43600; Patent Literature 2: Japanese Patent No. 4565168; Patent Literature 3: Japanese Patent Laid-Open No. 2-138856).
According to such a technique, exceedingly thinly focused charged particles can be allowed to be incident on the sample supporting film, suppressing a diffusion range of the charged particles to attain high resolution. Moreover, when a plurality of X-ray detectors are installed at various angles and positions below the sample supporting film (on its rear face side), inclined images depending on the installation angles can be obtained, affording as many inclined images as the number of the installed detectors in one charged particle beam scanning and also enabling a three-dimensional structure of the observation target sample to be reproduced based on these inclined images (Patent Literature 2).
As a sample supporting member (supporting film) used for such soft X-ray microscope observation, a silicon nitride film has been widely used. Since the silicon nitride film has excellent pressure resistance, it has been thought that even when the relevant silicon nitride film is provided in a window part of a sample-containing cell inside which is under atmospheric pressure, the usage of the sample-containing cell in the microscope apparatus inside which is under a vacuum suffers no difficulty at all. For example, according to the invention disclosed in Japanese Patent Laid-Open No. 6-180400 (Patent Literature 4), a sample cell in which two silicon nitride films are arranged opposite to each other with a predetermined interval is used, a recess for storing a sample solution is provided in the periphery of an X-ray transmission window, observation samples along with a solution are sealed between the silicon nitride films to be closed, and thus, X-ray microscope observation is performed.